yo usually obtained by the vacuum-fusion techniques. Also, the technique has been shown t o be accurate. Another factor of importance in evaluating the technique is the decreased cost of analysis. The cost per analysis (in this laboratory) by the platinum-flus technique is about 40% of that by the other vacuum-fusion tcrhniques. relative
Table IV.
Platinum-Flux Analysis of Unalloyed Titanium Standard Samples
Sample“ 1 2
3 4 5 6 7
8 9
LITERATURE CITED
( I ) Albrecht, TI7. M., Mallett, M. W., ANAL.CHEM. 26, 401 (1954).
(2) Bennett, S. J., Covington, L. C., Zbzd., 30, 363 (1958). (3) Derge, G., J . Metals 1, No. 10, 31 (1949). (4) Gregory, J. N., Mapper, D., Analyst 80. 230 (1955\. -~ (5) Hansen, W. R., Mallett, hl. W., Trzeciak, M. J., TML Rept. 89, Titanium Metallurgical Labbratory, Battelle Memorial Institute, Columbus, Ohio, Dee. 6, 1957. 161 Kroll. W. .J.. Schlerhton. A . W.. J . Electroihem. Sic. 93, 247 (1948). ( 7 ) Mallett, M. IT.,Griffith, C. B., Trans. A m . Sac. Metals 46, 375 (1954). (8) Smiley, W. G., ANAL. CmM. 27, 1098 (1955). (9) U. S. rlir Force Soecification Bull. 108A (May 6, 1955) ?superseding KO.
10
11 12 13 14 15 16
\
a
Sample Weight, Gram 0.245 0.238 0,187 0.257 0.268 0.212 0.214 0,278 0 234
0,228 0.219 0.197 0.231
Gaseous addition 0.143 0.143 0 145 0 139 0 141 0.142 0.140 0.156 0 297 0 294 0 280 0 303 0 290 0.29% 0 303 0 277 0.307
Oxygen, K t . % Estimated total ;inalysis 0 171 0 152 0 191 0 171 0 176 0 173 0 167 0 161 0.169 0 166 0 170 0 179 0 168 0 165 0 184 0 li,? 0 31J 0 324 0 321 0 278 0.30; 0 307 0 330 0 316 0 294 0.321
Oxygen Recovery,
%
89 112 102 97
98
105 98 95 97 87 100
96
93 99 0 330 0 309 93 0 324 0.296 $1I 0.189 17 0 334 0.196 0.315 91 18 0.250 0.283 0 310 0.301 97 Av. 97 Base material analyzed 0.028 weight 9c oxygen for samples 1 through 8, 0.027 for
0 . 177 0 196
samples 9 through 18.
108, Aug. 9, 1954), “Quality Requirements for Wrought Titanium and Titanium Alloys.” (10) Walter, D. I., ANAL. CHEw 22, 297 (1950).
!11) Wilkins, D. H., Fleischer, J. F., -4nal. Chim. Acta 15, 334 (1956).
RECEIVEDfor review July Accepted February 5, 1959.
14, 1958.
Rapid Colorimetric Method for Determining Glyoxal C. S. WISE, C. L. MEHLTRETTER, and J. W. VAN CLEVE Northern Utilization Research and Development Division, Agricultural Research Service,
F A rapid and selective spectrophotometric method for determining glyoxal in the presence of reducing sugars has been developed for use in studies on the production of glyoxal by the hydrolysis of periodate-oxdized starch. The method i s an extension of the work of Wanzlick and Lochel, who showed that 1 , I ’-3,3’-tetraphenyl-2,2’biimidazolidine, formed by reaction of glyoxal with dianilinoethane, produces a blue color upon heating with hydrochloric acid. A small aliquot containing about 1.2 mg. of glyoxal i s heated for 30 minutes with an alcoholic acid solution of dianilinoethane. After dilution to 100 ml. the absorbance is measured at 550 mp and is constant for at least 2 days. Formaldehyde, acetaldehyde, glucose, and erythrose do not interfere.
I
of the acid hydrolysis of periodate-oxidized starch, it was necessary to determine the amount of glyoxal produced as the reaction neared completion. A more facile colorimetric procedure was desired than the method of Dechary, Kun, and Pitot (Z), which utilizes 2,3-diaminophenazine and N A STUDY
requires several subsequent reactions to eliminate the effect of excps? reagent. Other methods for the quantitative estimation of glyoxal were either too time-consuming or not suited for use in an aqueous system containing reducing sugars (1, 3-5, 7 ) . *4 comparatively selective color reaction between glyoxal and 1,2-dianilinoethane in acid solution was described by Kanzlick and Lochel (8). The rolor produced by reaction with mineral acid of the intermediate l.l’-3,3’-tetrapheny1-2,2’-biimidazolidineformed by condensation of the above reagents was extremely sensitive to glyoxal concentration. The niethod was unaffected by reducing sugars and appearcd adequate for the quantitative detcrmination of glyoxal in solutions of highly hydrolyzed periodate ouystarch. The presence of a considtmlile quantity of unhydrolyzed ouystzrch interfered, through production of glyoxal during color formation n ith the acid rcagcnt. The method appears. lion-rwr. to be generally applicable for the a n a l ~ s i sof glyoxal in aqueous solutions. Thc reaction conditions of Wanzlick and Lochel were modified t o achieve optimum color development by increasing
U. S. Department o f Agriculture,
Peoria, Ill.
the ratio of dianilinoethane t o glJ-osal and conducting the reaction of 77” C. in an ethyl alcohol solution, under controlled acidity for a limited time. Absorbance was measured a t 550 mp and found to be constant for a t least 2 days. Formaldehyde, acetaldehyde, glucose, and erythrose did not interfere with the color reaction. APPARATUS AND REAGENTS
SPECTROPHOTOMETER, Colenian Junior Model 6ii, or similar anparatus. BOROSILICATE GLASSTUBES,18 X 150 mm., selected for uniformity in spectrophotometric meusurements. 1,2-DIANILINOETHANE DIHYDRCCHLO RIDE reagent (8). A stock solution was prepared by dissolving 1.3333 grams in 100 ml. of 9570 ethyl alcohol. ST.4NDARD SoLCTION O F GLYOXAL. A stock solution x i s made by weighing 0.1895 gram of the pure, crystalline monohydrate of glyoxal-sodium bisulfite (6) and diluting to 100 ml. with water. A 3-ml. aliiuot contains 1.161 mg. of glyoxal (0.02 mmole). PROCEDURE
Place in a test tube a 3-ml. aliquot of the unknonn gl\-oxal solution con. taining approximately 1.2 mg. of glyoxal. VOL. 31, NO. 7, JULY 1959
1241
T o this add 3 ml. of the alcoholic solution of 1,2-dianilinoethane dihydrochlo40 ml. of the reagent. The ride-i.e., reagent introduced is approximately 3.5 times the amount of dianilinoethane required for reaction with the glyoxal present to form 1,1’-3,3’-tetraphenyl2,2’-biimidazolidine. Add exactly 0.6 ml. of concentrated hydrochloric acid from a pipet, mix well, and heat in the water bath for 30 minutes a t 77” C. To prevent loss of ethyl alcohol cover the test tube with a small watch glass. After the heating period, pour the reaction mixture into a 100-ml. volumetric flask and dilute to the mark with 95% ethyl alcohol. Use part of
the diluting alcohol to wash the last of the blue liquid from the test tube into the flask. Place approximately 15 ml. of the solution in a test tube (matched tubes should be used) and measure the absorbance a t a wave length of 550 nip us. a blank made as described but containing water instead of the glyoxal solution. Use a known solution of glyoxal-sodium bisulfite monohydrate as a reference standard and run a t the same time. If the unknon n solution has approximately the absorbance of the standard, estimate the glyoxal concentration by using Beer’s law. A more precise procedure is to have tivo reference standards, betiveen which the concentration of glyoxal in the sample would lie. A cur1.e similar to that of Figure 1 can then be drawn for estimating the concentration of glyoxal in the sample.
Table 1. Determination of Glyoxal in Periodate Oxystarch Hydrolyzates
Recoverv of G1voxal.a 9/, Sodium Sample bisulfite No. complex Colorimetric 1 73.2 76.1 2 80.2 83.8 3 81.3 80.9 4 81.3 81.4 5 82.1 82.9 6 82.7 83.3 a Percentage of theoretical recovery of glyoxal from acid hydrolysis of periodate oxystarch a t 100fz oxidation level.
EXPERIMENTAL
The sensitivity of Wanzlick and Lochel’s procedure was greatly improwd by using an increased ratio of dianilinoethane reagent to glyoxal and by conducting the reaction in boiling ethyl alcohol instead of water. With these conditions standardized, optimum color formation depended on the acidity of the solution and time of reaction. Maximum color was produced when the amount of concentrated hydrovhloric acid used was between 0.G and 0.9 ml., as shown in Figure 2. Figure 3 shows that the absorbanre at 550 mp reached its peak in approximately 30 minutes and decreased a t a blow rate thereafter. Nearly maximum color development was obtained by heating between 20 and 40 minutes; hence, the time of reaction is not critical for obtaining accurate results. Formaldehyde, acetaldehyde, glucose, and erythrose had no appreciable effect on the color reaction. To determine this, 1.2 mg. of the interfering substance were added t o the standard 1.2 mg. of glyoxal. I n all cases the resulting increase in color was less than 1%. RESULTS A N D DISCUSSION
Figure 1. glyoxa I
I
0
The method is suitable for the analysis of aqueous solut.ions of glyoxal and has
Absorbance curve for
I
0.4
I
I
1
0.8 1.2 1.6 NIL. OF CONCENTRATED HCI
2.0
Figure 2. Effect of hydrochloric acid on absorbance
1242
ANALYTICAL CHEMISTRY
1
the advantages of simplicity and rapidity in determining glyoxal in the presence of reducing sugars and aldehydes such as formaldehyde and acetaldehyde. The reagent employed is readily prepared. Color formation appears to be specific for glyoxal and is extremely sensitive to glyoxal concentration. I n addition, absorbance at the wave length used for the analysis was stable for days. Ten replicates of the colorimetric determination m r e made on sample G and the standard deviation was found to be 1.27%. The method proved to be satisfactory for the determination of glyoxal produced by extensive acid hydrolysis of periodate oxidized starch. Table I shows a comparison of the results obtained by the colorimetric procedure and by the precipitation of the sodium bisulfite monohydrate complex of glyoxal. The average difference between the methods is less than 1.5%. ACKNOWLEDGMENT
The authors thank B. T. Hofreiter of this laboratory for preparing the 1,P-dianilinoethane dihydrochloride, and for calling attention to the work of Wanzlick and Lochel on the use of this reagent. LITERATURE CITED
(1) Ariysma, N., J. Biol. Chem. 77, 359 (1928). (2) Dechary, J. Sf.,Kun, E., Pitot, H. C., ANAL. CHEW26. 449 (1954). (3) Friedmann, T.’E., J.‘ Biol: Chem. 73, 331 (1927). (4)Gabrielsson, G., Samuelsson, O., Svensk Kem. Tidskr. 6 2 , 214 (1950). (5) Glasstone, S., Hickling, _. A.,. J. Chem.
. SOC.1936, 824.’
16) Ronzio. A. R.. Wawh. T. D.. Oraanic ‘ Synthesis, 24, 61 (19iii. ( 7 ) Salomaa, P., Acta ChenL. Scand. 10, 306 (1956). (8) Wanzlick, H. W., Lochel, W., Ber. deut. chem. Ges. 86, ( l l ) ,1463 (1953). ,
RECEIVEDfor review December 5, 1958 Accepted Fehruary 23, 1959. Division of Analytical Chemktry, 134th lfeeting, ACS, Chicago, Ill., September 1958. Mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.
I
I
20
0
I
40
r I
60
TIMt, MINUTES
Figure 3.
Effect of heating time on absorbance
0.6 ml. of concentrated hydrochloric acid used
